KR20130067266A - Therapeutic peptide composition and method - Google Patents

Abstract

Therapeutic peptide compositions include therapeutic peptides, such as insulin, together with alkyl N, N-disubstituted aminoacetates, such as dodecyl 2- (N, N-dimethylamino) propionate, and the like.

Description

Therapeutic Peptide Compositions and Methods {THERAPEUTIC PEPTIDE COMPOSITION AND METHOD}

Cross-reference to related application

This application claims the priority of US Provisional Application No. 61 / 343,815, filed May 4, 2010, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to therapeutic peptide compositions and methods of delivery of such compositions.

Peptides are mediators of biological function. The unique inherent properties of these peptides make the peptides attractive therapeutics because they exhibit relatively low toxicity as well as relatively high biological activity and specificity. However, in vivo therapeutic peptides have only some drawbacks such as relatively low stability, sensitivity to enzymatic degradation, relatively poor tumor penetration in cancer treatment, and the like.

Therapeutic peptides are viable alternatives to other biopharmaceuticals, but their stability and in vivo half-life continue to be a problem.

The therapeutic peptide composition of the present invention embodying the present invention provides a depot-like effect when administered to a patient, thereby prolonging the treatment period of the administered peptide several times.

The therapeutic peptide composition of the present invention comprises a therapeutic peptide and an alkyl N, N-disubstituted aminoacetate, if necessary, together with a physiologically acceptable carrier. In any given case, the dosage and dosage form of the therapeutic peptide depends on the condition being treated, the particular therapeutic peptide used to treat the condition, as well as the desired route of administration.

Compositions comprising insulin and dodecyl 2- (N, N-dimethylamino) propionate hydrochloride are particularly suitable for controlling blood glucose levels in diabetic patients.

In the figure,
1 is a graph depicting blood glucose levels in normal mice over a period of time after administration of insulin in saline and in dodecyl 2- (N, N-dimethylamino) propionate;
FIG. 2 is a graph of serum levels versus time of Biot-Rituxan in samples from hamsters injected with a subcutaneous dose in saline and in dodecyl 2- (N, N-dimethylamino) propionate. ;
3 shows plasma levels of liraglutide in samples from mice injected with a subcutaneous dose of clinical formulation, with and without dodecyl 2- (N, N-dimethylamino) propionate. Graph of time;
FIG. 4 is a graph showing blood glucose levels in mice subcutaneously injected with insulin 2.5 IU / kg in 0.9% saline and 20% DDAIP.HCl (pH 8.5) in water over an 8-hour period;
FIG. 5 is a graph depicting blood glucose levels in mice subcutaneously injected with insulin 2.5 IU / kg in 0.9% saline and 20% DDAIP.HCl (pH 8.5) in water over a 26-hour period, wherein mice were injected after 8 hours. Access to food.

Definition of Terms

As used herein and in the appended claims, the term "peptide" means any compound that contains two or more amino acid residues linked by an amide bond formed from an amino group of an amino acid residue adjacent to a carboxyl group of one amino acid residue. It is also referred to as. Amino acid residues can have L-form as well as D-type, and can also be natural, synthetic, linear as well as cyclic. In addition, within the term "peptide" as used in this specification and the appended claims, a C-terminally linked peptide (tandem repeat) or a C-terminus-coupled peptide (parallel) Also included are peptide dimers and polypeptides, which can be repeated).

The term "therapeutic peptide" as used in this specification and the appended claims refers to a bioactive peptide having therapeutic utility. Exemplary categories of therapeutic peptides suitable for practicing the present invention are hormones, extracellular matrix (ECM) peptides, enzymes, cytokines and the like.

Physiologically Acceptable Carrier: As used herein, the term "physiologically acceptable carrier" refers to a diluent, adjuvant, excipient, or other vehicle with which the therapeutic peptide is administered. Such carriers are sterile liquids such as water and oils such as petroleum derived oils, animal derived oils, plant derived oils or synthetic derived oils such as peanut oil, soybean oil, mineral oil, sesame oil, tocophenols, polyethylene, etc. Glycol, glycerine, propylene glycol, or other synthetic solvents. Water is the preferred carrier when the therapeutic peptide is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injection solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, glycol, water, ethanol or any compound found in Handbook of Pharmaceutical Excipients (4 ^th edition, Pharmaceutical Press). Small amounts of wetting or emulsifying agents, or pH buffers such as acetate, citrate or phosphate may also be present. Moreover, antibacterial agents, such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium hydrogen sulfite; Chelating agents such as ethylenediaminetetraacetic acid; And toxic modifiers such as sodium chloride or dextrose.

A therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount that will have a desirable therapeutic effect when administered to a subject in view of the nature and severity of the disease or condition of the particular subject, For example, an amount that cures, prevents, inhibits, or at least partially arrests or partially prevents, a target disease or condition.

Exemplary hormones include insulins such as human insulin, bovine insulin, porcine insulin, biosynthetic human insulin (Humulin®) and the like, somatostatin, vasopressin, calcitonin, estrogen, progestin, testosterone, glucagon, glucagon-like Peptides (GLP-1) and analogs thereof, such as liraglutide (VICTOZA®) and the like. Monoclonal antibodies can be of various types, such as mouse, chimeric, humanized or human. Exemplary chimeric monoclonal antibodies include Rituximab (RITUXAN®), Cetuximab (ERBITUX®), Infliximab (REMICADE®), Basiliximab (SIMULECT®), and the like. Exemplary humanized monoclonal antibodies include Trastuzumab (Herceptin® (HERCEPTIN®), Palivizumab (SYNAGIS®)), Epalizumab (Raptiva). Exemplary human monoclonal antibodies include Adalimumab (HUMIRA®), Panitumumab (VECTIBIX®). ) And so on.

Exemplary ECM peptides are fibronectin, vitronectin, tenascin and the like.

Exemplary enzymes are glucocerebrosidase, rhDNase, hyaluronidase, urokinase, alpha galactosidase, beta galactosidase, and the like.

Exemplary cytokines include α, β and γ interferons, lymphokines such as interleukin-2, interleukin-6, and the like, human granulocyte colony stimulating factor (G-CSF) such as filgrastim, granulocyte macrophage colony stimulation Factor (GM-CSF), recombinants such as molar grasteam, Sagra mos tim (LEUKINE®) and the like.

Alkyl N, N-disubstituted aminoacetates suitable for the purposes of the present invention are represented by the formula:

N is an integer having a value ranging from about 4 to about 18; R is a member selected from the group consisting of hydrogen, C ₁ to C ₇ alkyl, benzyl and phenyl; R ₁ and R ₂ are members selected from the group consisting of hydrogen and C ₁ to C ₇ alkyl; R ₃ and R ₄ are members selected from the group consisting of hydrogen, methyl and ethyl.

Preferred alkyl (N, N-disubstituted amino) -acetates are C ₄ to C ₁₈ alkyl (N, N-disubstituted amino) -acetates and C ₄ to C ₁₈ alkyl (N, N-disubstituted amino) -propionates as well Pharmaceutically acceptable salts and derivatives thereof. Exemplary specific alkyl-2- (N, N-disubstituted amino) -acetates are dodecyl 2- (N, N-dimethylamino) -propionate (DDAIP):

And dodecyl 2- (N, N-dimethylamino) -acetate (DDAA):

.

Alkyl-2- (N, N-disubstituted amino) -acetates are known. For example, dodecyl 2- (N, N-dimethylamino) -propionate (DDAIP) is available from Steroids, Ltd (Chicago, Ill.). Alkyl-2- (N, N-disubstituted amino) -alkanoate is also described in US Pat. No. 4,980,378 (Wong et al., Incorporated herein by reference to the extent that it does not contradict). Such as more readily available compounds. As described herein, alkyl-2- (N, N-disubstituted amino) -acetates are readily prepared through two step synthesis. In the first step, long chain alkyl chloroacetates are prepared by reaction of the corresponding long chain alkanols with chloromethyl chloroformate, typically in the presence of a suitable base such as triethylamine, in a suitable solvent such as chloroform and the like. This reaction can be expressed as follows:

Wherein n, R, R ₁ , R ₂ , R ₃ and R ₄ are defined as above. The reaction temperature may be selected from about 10 ° C. to about 200 ° C. or reflux, but room temperature is preferred. The use of a solvent is optional. If a solvent is used, a wide range of organic solvents can be selected. The choice of base is likewise not important. Preferred bases include tertiary amines such as triethylamine, pyridine and the like. The reaction time generally extends from about 1 hour to 3 days.

In the second step, the long chain alkyl chloroacetate is condensed with the appropriate amine according to the following scheme:

Wherein n, R, R ₁ , R ₂ , R ₃ and R ₄ are defined as above. Excess amine reactant is typically used as a base and this reaction is suitably carried out in a suitable solvent such as ether. This second step is preferably carried out at room temperature, but the temperature may vary. The reaction time can typically vary from about 1 hour to several days. Conventional purification techniques can be applied to prepare the esters obtained for use in pharmaceutical compounds.

The amount of alkyl N, N-disubstituted aminoacetates, such as DDAIP, etc., present in the therapeutic peptide composition can vary, depending in part on the route of administration as well as the particular peptide to be administered.

The invention is further illustrated by the following examples.

Example 1 Delivery of Insulin in DDAIP

Insulin (bovine insulin, manufactured by Sigma) at a concentration of 3.2 IU / ml in DDAIP (free base) (n = 3) and in phosphate buffered saline (n = 2) was administered subcutaneously (single dose, 0.4 IU). /mouse). Blood glucose levels after infusion were monitored at various time intervals before and after administration. The results are shown in FIG. 1, which shows that DDAIP has a depot-like dissociation effect on therapeutic peptides such as subcutaneously delivered insulin and the like.

Example 2: Delivery of Monoclonal Antibodies in DDAIP

Biotinylated Rituximab (Biot-Rituxan®) (9.4 mg / ml), DDAIP Free Base (4.9 or 369% w / v) and Polyoxyethylene in 0.1M Phosphate Buffer at pH 5.5 or 7.4 20) A formulation of sorbitan monolaurate (Tween® 20) (5% w / v) was prepared and administered subcutaneously (SC) to a group of three hamsters as a single dose. The subcutaneous dose was 10 mg / kg. Three hamsters from another group were administered subcutaneously with 10 mg / kg of Biot-Rituxan® in saline.

Blood samples (100 mL) were taken from the animals in red serum tubes kept on ice. The collected sample was processed by centrifugation at about 3,000 RPM for about 10 minutes. The resulting supernatant serum was transferred to a polypropylene tube, placed in dry ice and frozen, and stored at −80 ° C. until analysis. Cell fractions obtained by centrifugation were discarded.

The data obtained are shown in FIG. 2. Formulations at pH about 7.4 have an area under the curve of about 21% for compositions containing about 4.9 parts by weight of DDAIP and about 46% for compositions containing 36.9 parts by weight of DDAIP compared to Biot-Ritux®® in saline ( AUC).

Example 3: Delivery of Glucagon-Like Peptide 1 (GLP-1) Analogues in DDAIPHCl

Male ICR (CD-1) mice were obtained from Harlan, USA. Mice were approximately 7 weeks old, had an average body weight of about 36 grams, and were freely provided with food and water. The experimental group classification is shown in Table 1 below.

Liraglutide (Victoza®) is combined with 5% w / v DDAIP hydrochloride (DDAIP.HCl), and the resulting clinical formulation is a single dose, administered subcutaneously (SC) immediately to the group of 21 mice. It was. The dose administered subcutaneously was 600 micrograms (mg) / mouse in 100 microliters (μl). Twenty one mice in the other group were subcutaneously administered a clinical formulation of liraglutide without DDAIP.HCl. Groups of three mice were not treated and used as baseline controls.

Experiment group classification Mouse group cure Per mouse
Volume Per mouse
volume Route therapy Per point
Blood drawing One
(n = 21) Liraglutide (Victoza®) Clinical Formulations
Annotation (b) 600 µg 100 μl SC 1 time n = 3
Annotation (a) 2
(n = 21) Liraglutide (Victoza®) Clinical Formulation + 5% w / v DDAIPHCl 600 µg 100 μl SC 1 time n = 3
Annotation (a) 3
(n = 3) none
(Baseline control) N / A N / A N / A N / A n = 3
Time = 0 Note to Table 1 : (a) : The time points were 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours and 24 hours after dosing.
Note to Table 1 (b) : Clinical Formulation Each 1 ml contains 6 mg of liraglutide. Each prefilled pen contains 18 mg of liraglutide (anhydrous free salt) and the following inactive ingredients: disodium phosphate dianhydride 1.42 mg, propylene glycol 14 mg, phenol 5.5 mg and sufficient water 3 ml of the solution to be contained.

Blood Collection:

At each time point indicated in Table 1, groups of mice (n = 3 per data point) were anesthetized with isoflurane before terminal cardiac blood collection performed with a 25 gauge needle. Blood samples were collected in K ₂ EDTA tubes and centrifuged for 10 minutes at a temperature of 10,000 rpm, 4 ° C. Plasma samples were collected and stored at −80 ° C. until analyzed by LCMS-MS.

Results and Conclusion:

FIG. 3 shows the pharmacokinetic profiles following subcutaneous treatment with liraglutide in groups 1 and 2. FIG. Table 2 below details the individual pharmacokinetic parameters calculated with PK Solutions 2.0 software (Summit Research Services, Montrose, Colorado). Compared with treatment with liraglutide in the clinical formulation alone, the addition of 5% w / v DDAIP.HCl resulted in lower C _max values and AUC values. In contrast, administration with 5% w / v DDAIP.HCl resulted in higher t _1/2 values, Vd values and MRT values. In conclusion, this study shows that therapeutically relevant systemic levels of liraglutide can be achieved when administered with a 5% w / v DDAIPHCl formulation. In addition, DDAIP-containing formulations provide a clinical benefit as these levels can maintain a better distribution (Vd) over a longer period of time than the clinical formulation (t _1/2 , MRT).

LCMS-MS Analysis of Samples Formulation Parameter (unit) (Group 1)
Lira glutide
(Victor (registered trademark)) Cmax (ng / ml) 81,560 Tmax (hr.) 4.0 T1 / 2 (hr.) 15.7 Vd (ml) 201 CL (ml / hr) 18.1 AUC (0-24) (ng-hr / ml) 1,128,000 AUC (0-∞) (ng-hr / ml) 1,323,000 Average residence time (MRT, hr.) 13.1 (Group 2)
With 5% w / v DDAIPHCl
Lira glutide
(Victor (registered trademark)) Cmax (ng / ml) 20,370 Tmax (hr.) 4.0 T1 / 2 (hr.) (Extrapolated) 25.3 Vd (ml) 1,024 CL (ml / hr) 36.3 AUC (0-24) (ng-hr / ml) 356,900 AUC (0-∞) (ng-hr / ml) 661,800 Average residence time (MRT, hr.) 30.1

Example 4 Effect of Insulin in DDAIPHCl on Blood Glucose Levels

Insulin (2.5 IU / kg) was combined with 20% w / v DDAIPHCl and 0.9% saline in water (pH 8.5) and male C57BL / 6J mice (n = 6, Jackson Labs., Mean weight). 33.4 g) was administered subcutaneously (SC dose, 5 mL / kg). For comparison, insulin in 0.9% saline (2.5 IU / kg) without DDAIP.HCl was also administered subcutaneously to mice (n = 6). Mice were initially administered with insulin in the presence of food, then food was discontinued after SC administration and food was reintroduced into mice 8 hours after infusion. Water was optionally available at all times.

Blood glucose levels were measured prior to dosing and then monitored after infusion at various time intervals using a handheld blood glucose meter (ACCU-CHEK®, Aviva). Blood samples were obtained from the tail vein at initial (T ₀ ) and at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours and 26 hours after administration.

Changes in blood glucose levels are shown in FIG. 4 for mice subcutaneously administered 2.5 IU / kg insulin over 6 hours prior to granting access to food, and also in FIG. 5 for mice over the entire 26-hour period. It is.

The obtained data presented in FIG. 4 showed that over a period of 0 to 6 hours, the AUC value obtained by insulin in saline was 778 ± 53 and the AUC value obtained by insulin in saline with 20% DDAIPHCl (both tails). t-test indicates 621 ± 29) at a confidence level of P = 0.0256. The data obtained in FIG. 5 show that over a period of 0 to 26 hours, the AUC value obtained by insulin in saline was 3,453 ± 170 and the AUC value obtained by insulin in saline with 20% DDAIPHCl (both tails). t-test indicates 2,567 ± 80) at a confidence level of P = 0.0008.

The results showed that the AUC values at 0-6 hours and 0-24 hours were lower than those in saline with 20% w / v DDAIPHCl (pH 8.5) than in mice injected with insulin in saline without DDAIPHCl. Significantly lower in the injected mice. The ability of 20% DDAIP.HCl to increase the efficiency of SC administered insulin is demonstrated by greater reduction of blood glucose levels at individual time points and by lower AUC values throughout the study period.

Parenteral delivery is preferred for the compositions of the present invention. Particularly preferred is subcutaneous delivery to maximize the depot effect.

The compositions of the present invention may be formulated as dosage forms, such as solutions, lyophilized powders, capsules, tablets, liposomes, etc., depending on the particular therapeutic peptide and the desired route of administration.

Suitable dosage forms for therapeutic peptide compositions include parenteral solutions, capsules, tablets, suppositories, gels, creams, and the like.

The above description and examples are intended to be illustrative and should not be treated as limitations. Other variations are possible within the spirit and scope of the invention, and those skilled in the art will readily provide them.

Claims (14)

A composition comprising a therapeutic peptide, an alkyl N, N-disubstituted aminoacetate, and a physiologically acceptable carrier. The composition of claim 1, wherein the therapeutic peptide is insulin and the alkyl N, N-disubstituted aminoacetate is represented by the formula:
[Chemical Formula]

N is an integer having a value ranging from about 4 to about 18; R is a member selected from the group consisting of hydrogen, C ₁ to C ₇ alkyl, benzyl and phenyl; R ₁ and R ₂ are members selected from the group consisting of hydrogen and C ₁ to C ₇ alkyl; R ₃ and R ₄ are members selected from the group consisting of hydrogen, methyl and ethyl. The composition of claim 1, wherein the therapeutic peptide is insulin and the alkyl N, N-disubstituted aminoacetate is dodecyl 2- (N, N-dimethylamino) propionate. The composition of claim 1, wherein the therapeutic peptide is insulin and the alkyl N, N-disubstituted aminoacetate is dodecyl 2- (N, N-dimethylamino) propionate hydrochloride. The composition of claim 1, wherein the therapeutic peptide is a monoclonal antibody. The composition of claim 5, wherein the monoclonal antibody is Rituximab. The composition of claim 5, wherein the monoclonal antibody is Raptiva. The composition of claim 1, wherein the therapeutic peptide is a cytokine and the alkyl N, N-disubstituted aminoacetate is dodecyl 2- (N, N-dimethylamino) propionate. The composition of claim 1, wherein the therapeutic peptide is a cytokine and the alkyl N, N-disubstituted aminoacetate is dodecyl 2- (N, N-dimethylamino) propionate hydrochloride. 10. The composition of claim 9, wherein the cytokine is human granulocyte colony stimulating factor (G-CSF). 10. The composition of claim 9, wherein the cytokine is a granulocyte macrophage colony stimulating factor (G-CSF) recombinant. The method of claim 1, wherein the therapeutic peptide is selected from the group consisting of glucagon-like peptide (GLP-1) and analogs thereof, and the alkyl N, N-disubstituted aminoacetate is dodecyl 2- (N, N-di Methylamino) propionate. 13. The composition of claim 12, wherein said alkyl N, N-disubstituted aminoacetate is dodecyl 2- (N, N-dimethylamino) propionate hydrochloride. The composition of claim 13, wherein the therapeutic peptide is liraglutide.

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